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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 07 January 2010 and 15 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The department of health of the government of the United Kingdom
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media: RPMI 1640
Properly maintained: yes
Periodically checked for Mycoplasma contamination: yes
Periodically checked for karyotype stability: no
Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
A maximum dose level of 5000 µg/mL, the maximum recommended dose level, was used.

Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 142314732109252 at 400 µg/mL and 150 µg/mL for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/mL was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
Vehicle used: RPMI 1640 without serum (R0)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without S9 mix, and 24 hours without S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates containing TFT selective medium. The microtitre plates were incubated for 10 to 14 days.
- Selection time: 10 - 14 days

SELECTION AGENT: 4 µg/mL 5-trifluorothymidine (TFT)

STAIN: MTT (2.5 mg/mL in PBS)

NUMBER OF REPLICATIONS: duuplicates each in two independent experiments in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.


Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
IMF has to exceed 126 E6 in order to be considered as positive.
However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 3750 µg/mL onward for 4h treatment + S9 and 24h treatment -S9; at 5000 µg/mL for 4 hour treatment - S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes when test material was dosed into media
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Evaporation from medium: no
- Water solubility: slightly soluble
- Precipitation: at and above 312.5 µg/mL
- Definition of acceptable cells for analysis: Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick.

RANGE-FINDING/SCREENING STUDIES: The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/ml for all three of the exposure groups. In the 4-hour exposure group in the absence of metabolic activation there were no marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle control. However, a modest reduction was observed in the 4-hour exposure group in the presence of metabolic activation, and a much more marked reduction was observed in the 24-hour exposure group. A precipitate of the test material was observed at and above 19.53 µg/ml in all three of the exposure groups. In the subsequent mutagenicity test the maximum dose level for all three of the exposure groups was the maximum recommended dose level of 5000 µg/ml.

HISTORICAL CONTROL DATA
- Positive historical control data: Mutation frequencies ranged from 466.26 to 2223.55
- Negative (solvent/vehicle) historical control data: Mutation frequencies ranged from 65.97 to 184.74

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Any other information on results incl. tables

Please see Attached "Tables 1 to 10"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Applicant's summary and conclusion

Conclusions:
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.